Aminoglycoside antibiotics: A-site specific binding to 16S

Abstract

The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S 27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S 27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S 27 alone illustrated both single-stranded 16S 27 and double-stranded (16S 27) 2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S 27, while either one or two bind to (16S 27) 2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S 27 and (16S 27) 2 complexes. These studies found 16S 27 in a hairpin conformation while (16S 27) 2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S 27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S 27) 2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S 27 complexes.